Urokinase (E.C. 3.4.21.31) is a serine protease which activates plasminogen to plasmin. The protein is synthesized in a variety of tissues including endothelium and kidney, and is excreted in trace amounts into urine. Purified urokinase exists in two active forms, a high molecular weight form (HUK; approximately 50 K) and a low molecular weight form (LUK; approximately 30 K). The entire amino acid sequence of both human forms has been determined (1,2,3). LUK has been shown to be derived from HUK by a proteolytic clip after lysine 135; this clip releases the first 135 amino acids from HUK (1). Conventional wisdom has held that HUK or LUK must be converted to proteolytically active forms by the proteolytic hydrolysis of a single chain precursor, also termed prourokinase, between lysine 158 and isoleucine 159 to generate a two-chain activated form (which continues to correspond to either HUK or LUK). The proteolytically active urokinase species resulting from this hydrolytic clip contain two amino acid chains held together by a single disulfide bond. The two chains formed by the activation clip are termed the A or A.sub.1 chains (HUK or LUK, respectively), and the B chain containing the protease domain of the molecule.
Urokinase has been shown to be an effective thrombolytic agent. However, since it is produced naturally in trace quantities the cost of the enzyme is high for an effective dosage. Urokinase has been produced in recombinant cell culture, and DNA encoding urokinase is known together with suitable vectors and host microorganisms (3,8).
As noted above, it has been believed that plasminogen activators exist as proteolytically inactive zymogens that must be "activated" by proteolysis before the enzyme can act upon plasminogen to commence the fibrinolytic cascade (4,5). While it has been observed that the urokinase single-chain proenzyme demonstrates high levels of activity on zymographic and fibrinolytic procedures (4,6), the fact that the proenzyme also exerts only low amidolytic activity on low molecular weight synthetic polypeptide substrates has led to the conclusion that traces of contaminating active (two-chain) urokinase in the proenzyme preparations, or traces of plasmin in the plasminogen used in the fibrin plate assays, accounted for the fibrinolytic activity of prourokinase (4). This in turn would compel the conclusion that single-chain urokinase must be converted to the two-chain form in order to cleave plasminogen and initiate fibrinolysis in vivo.
The role of urokinase in clot lysis in vivo is complicated. Prourokinase now is believed to interact with an inhibitor in plasma. Fibrin is postulated to release this inhibitor, whereupon prourokinase is released for action on plasminogen (7). It is unclear whether removal of the inhibitor alone is sufficient to initiate plasminogen hydrolysis, or whether release from the inhibitor simply facilitates conventional urokinase activation to the two-chain form The urokinase domain bound by the inhibitor is unknown, nor is the binding mechanism known. A further complicating hypothesis attributes a fibrin-binding capability of urokinase to the HUK species, in particular to a region called a "kringle" located within about residues 49 to 132 (8). The relationship of this hypothesis to the inhibitor postulate, and their comparative merit, remains unresolved.
A major impediment to the use of two-chain urokinase for the treatment of blood clots is that the two-chain form is apparently not bound by the putative inhibitor of prourokinase. If two-chain urokinase is administered peripherally it is therefore capable of activating plasminogen at any point within the circulatory system, thereby leading to undesirable side effects. Two-chain urokinase generated by plasmin hydrolysis of single-chain urokinase at the clot site enters circulation with the same adverse side effects, in particular systemic fibrinogenolysis and depletion of .alpha.2 anti-plasmin. These side effects hamper proper thrombogenesis in vivo. An improved fora of single-chain urokinase is needed (a) which is capable of binding either fibrin or the postulated inhibitor, i.e., which in the end functions substantially the same as native prourokinase with respect to plasminogen activation at clot sites; (b) which is resistant to proteolytic digestion in particular to conversion to the two-chain form; and (c) which exhibits minimal or no antigenicity in patients to whom it is administered.